Neonatal stroke often leads to mental retardation, cerebral palsy, or epilepsy. The injurious mechanisms of hypoxia and ischemia in immature brain are thought to differ from those in mature brain, therefore requiring age-appropriate therapeutic interventions. Rapid activation of resident microglia upon reperfusion following cerebral ischemia in immature animals is one of the mechanisms that propagate reperfusion injury. Activation of microglia at least in part depends on the mitogen-activated protein kinase (MAP kinase) p38. We hypothesize that reperfusion injury in the neonatal brain is mediated by microglial activation via activation of MAP kinase p38. We will use our transient focal ischemia-reperfusion model in neonatal rats to explore the role of MAP kinase p38 in neuroprotection. Specifically, we will determine whether: 1) MAP kinase p38 contributes to the acute injury (Aim 1); 2) injurious effects of activated microglia are mediated by MAP kinase p38 (Aim 2); and 3) pharmacological reduction of oxidative stress attenuates microglial activation and protects the neonatal brain (Aim 3). We will determine whether pharmacological inhibition of MAP kinase p38 attenuates injury, both neuronal necrosis and apoptosis, by decreasing the magnitude of cytokine and nitric oxide production in activated microglia. We will determine whether lessening the oxidative stress attenuates p38-dependent activation of microglia and injury. Our ability to map the evolving ischemic core and penumbra using diffusion-weighted magnetic resonance imaging (DW-MRI) will allow us to follow injury evolution in treated and non-treated ischemic pups. In cultured microglia, we will explore signaling mechanisms of p38-mediated activation of microglia (Aim 4), in particular, the contribution of two signal transduction cascades that regulate p38, MBKK1- and ASK1-dependent modules. We will determine whether p38-mediated microglial activation exacerbates hypoxic injury to cultured primary neurons (Aim 4). The study of a pro-inflammatory pathway using a new model in the neonate and the proposed multidisciplinary approach will advance knowledge about the role of oxidative and pro-inflammatory mechanisms of reperfusion injury in neonatal stroke.